The present invention provides mutant presenilin 1 and presenilin 2 polypeptides and polynucleotides encoding the polypeptides and methods for their production by recombinant and PCR techniques are disclosed. Methods for utilizing the mutant polypeptides in cell based and in-vitro assays for inhibitors of activity are also disclosed.
Alzheimer""s disease was originally thought to be a rare disorder primarily affecting only people under the age of 65. It is now recognized as the most common form of dementia, and alone is responsible for about 50% of all dementias; an additional 15-20% of dementias have combined Alzheimer""s and vascular pathology.
The prevalence of the Alzheimer""s is directly related to age. It can occur in the fourth decade of life but is extraordinarily rare at this age. The prevalence then increases logarithmically with each succeeding decade. Over the age of 85 at least one person in four is afflicted. Because persons over the 85 form the rapidly growing portion of the population Alzheimer""s disease represents a major health problem. Zigmond, et.: Fundamental Neuroscience, Academic Press, 1999.
Alzheimer""s disease is thought to be initiated by the deposition of amyloid plaque in cortex and hippocampus. The material deposited in plaque is proteinaceous. It consists primarily of the amyloid xcex2-peptide (Axcex2), a peptide of 39-43 amino acids which is derived from a larger precursor, the amyloid peptide precursor (APP), through the action of specific proteases. APP is a large, type-I transmembrane protein of 695-770 amino acids that is expressed by a variety of cell types including neurons, glia and somatic cells. The cleavage of Axcex2 from APP is accomplished by the action of two proteolytic activities commonly denoted as beta-secretase (Asp2) and gamma-secretase. Processing at the xcex3-secretase site is somehow dependent on presenilin-1 (as it does not occur in PS1 null embryonic neurons grown in culture, DeStrooper et al., 1997), but the protease responsible has not been identified. Deletion of the PS1 gene in mice greatly reduces gamma secretase activity. With less than 5% of the APP made by the cell processed through the amyloidogenic pathway to Axcex2. DeStrooper (1998); Qian (1998).
A causative role for Axcex2 peptide in Alzheimer""s disease is supported by genetic studies of familial, early-onset Alzheimer""s disease in which inheritance follows an autosomal dominant mode of transmission. In such patients, genetic forms of Alzheimer""s disease have been associated with mutations in the APP gene (Groate et al., 1991; Mullan et al. 1992), and two related genes, presenilin-1 (PS-1; Sherrington et al., 1995) and presenilin-2 (PS-2; Levy-Lahad et al., 1995; Rogaev et al., 1995). Mutations in all three genes alter production of the Axcex2 peptide in specific ways.
PS1 and PS2 mutations subtly increase the production of Axcex21-42 peptide as compared to the Axcex21-40 peptide (e.g., Citron et al., 1997), Mehta et al. (1998), Murayama et al. (1999), Xia et al. (1997). Axcex21-42 is generally recognized as being more toxic to cells than Axcex21-40. 
Because PS1 and PS2 are intimately involved with the processing of APP both genes are attractive targets for drug screening in which aberrant APP processing is a causative or exacerbating factor. It has been postulated that both presenilin 1 and presenilin 2 have some intrinsic protease activity but this activity is so weak that designing a method of screening test agents which inhibit the intrinsic activity is problematic. The invention provides mutant presenilin 1 and presenilin 2 with enhanced proteolytic activities suitable for high throughput screening.
Literature Cited
1. Citron, M. et al. Mutation of the beta-amyloid precursor protein in familial Alzheimer""s disease increases beta-protein production. Nature 360, 372-374 (1992).
2. Citron, M.; Westaway, D.; Xia, W.; Carlson, G.; Diehl, T.; Levesque, G.; Johnson-Wood, K.; Lee, M.; Seubert, P.; Davis, A.; Kholodenko, D.; Motter, R.; Sherrington, R.; Perry, B.; Yao, H.; Strome, R.; Lieberburg, I.; Rommens, J.; Kim. S.; Schenk, D.; Fraser, P.; St George Hyslop, P.; Selkoe, D. J. : Mutant presenilins of Alzheimer""s disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice. Nature Med. 3: 67-72, 1997.
3. De Strooper B, Saftig P, Craessaerts K, Vanderstichele H, Guhde G, Annaert W, Von Figura K, Van Leuven F. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature. Jan. 22, 1998 ;391 (6665):387-90
4. Goate, A.; Chartier-Harlin, M.-C.; Mullan, M.; Brown, J.; Crawford, F.; Fidani, L.; Giuffra, L.; Haynes, A.; Irving, N.; James, L.; Mant, R.; Newton, P.; Rooke, K.; Roques, P.; Talbot, C.; Pericak-Vance, M.; Roses, A.; Williamson, R.; Rossor, M.; Owen, M.; Hardy, J. : Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer""s disease. Nature 349: 704-706, 1991
5. Levy-Lahad, E.; Wasco, W.; Poorkaj, P.; Romano, D. M.; Oshima, J.; Pettingell, W. H.; Yu, C.; Jondro, P. D.; Schmidt, S. D.; Wang, K.; Crowley, A. C.; Fu, Y.-H.; Guenette, S. Y.; Galas, D.; Nemens, E.; Wijsman, E. M.; Bird, T. D.;Schellenberg, G. D.; Tanzi, R. E. : Candidate gene for the chromosome 1 familial Alzheimer""s disease locus. Science 269: 973-977, 1995.
6. Mehta N D, Refolo L M, Eckman C, Sanders S, Yager D, Perez-Tur J, Younkin S, Duff K, Hardy J, Hutton M. Increased Abeta42(43) from cell lines expressing presenilin 1 mutations. Ann.Neurol. 1998 Feb; 43(2):256-8.
7. Murayama O, Tomita T, Nihonmatsu N, Murayama M, Sun S, Honda T, Iwatsubo T, Takashima A. Enhancement of amyloid 42 secretion by 28 different presenilin 1 mutations of familial Alzheimer""s. Neuroscience Letters 1999 April; 265 (1):61-63.
8. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B., Lannfelt, L. A pathogenic mutation for probable Alzheimer""s disease in the APP gene at the N-terminus of beta amyloid. Nat Genet. 1, 345-347 (1992).
9. Rogaev, E. I.; Sherrington, R.; Rogaeva, E. A.; Levesque, G.; Ikeda, M.; Liang, Y.; Chi, H.; Lin, C.; Holman, K.; Tsuda, T.; Mar, L.; Sorbi, S.; Nacmias, B.; Placentini, S.; Amaducci, L.; Chumakov, I.; Cohen, D.; Lannfelt, L.; Fraser, P. E.; Rommens, J. M.; St George-Hyslop, P. H. : Familial Alzheimer""s disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer""s disease type 3 gene. Nature 376: 775-778, 1995.
10. Sherrington, R.; Rogaev, E. I.; Liang, Y.; Rogaeva, E. A.; Levesque, G.; Ikeda, M.; Chi, H.; Lin, C.; Li, G.; Holman, K.; Tsuda, T.; Mar, L.; Foncin, J.-F.; Bruni, A. C.; Montesi, M. P.; Sorbi, S.; Rainero, I.; Pinessi, L.; Nee, L.; Chumakov, I.; Pollen, D.; Brookes, A.; Sanseau, P.; Polinsky, R. J.; Wasco, W.; Da Silva, H. A. R.; Haines, J. L.; Pericak-Vance, M. A.; Tanzi, R. E.; Roses, A. D.; Fraser, P. E.; Rommens, J. M.; St George-Hyslop, P. H. : Cloning of a gene bearing mis-sense mutations in early-onset familial Alzheimer""s disease. Nature 375: 754-760, 1995.
11. Suzuki, N., et al. An increased percentage of long amyloid beta protein secreted by amilial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336-1340 (1994).
12. Qian S, Jiang P, Guan X M, Singh G, Trumbauer M E, Yu H, Chen H Y, Van de Ploeg L H, Zheng H. Mutant human presenilin 1 protects presenilin 1 null mouse against embryonic lethality and elevates Abeta 1-42/43 expression. Neuron. 1998 Mar; 20(3):611-7.
13. Xia W, Zhang J, Kholodenko D, Citron M, Podlisny M B, Teplow D B, Haass D, Seubert P, Koo E H, Selkoe D J. Enhanced Production and Oligomerization of the 42-residue Amyloid-Protein by Chinese Hamster Ovary Cells Stably Expressing Mutant Presenilins J. Biol. Chem. 1997;272:7977-7982.
14. Zigmond, M. J, Bloom, F. E., Landis, S. C., Roberts, J. L., Squire, L. R.:
Fundamental Neuroscience, Academic Press, 1999.
15. Li Y M, Xu M, Lai M T, Huang Q, Castro J L, DiMuzio-Mower J, Harrison T, Lellis C, Nadin A, Neduvelil J G, Register R B, Sardana M K, Shearman M S, Smith A L, Shi X P, Yin K C, Shafer J A, Gardell S J. Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1. Nature. Jun. 8, 2000; 405(6787):689-94.
16. Rishton G M, Retz D M, Tempest P A, Novotny J, Kahn S, Treanor J J, Lile J D, Citron M. Fenchylamine sulfonamide inhibitors of amyloid beta peptide production by the gamma-secretase proteolytic pathway: potential small-molecule therapeutic agents for the treatment of Alzheimer""s disease. J Med Chem. Jun. 15, 2000; 43(12):2297-9.
17. Tajima K, Babich S, Yoshida Y, Dantes A, Strauss J F 3rd, Amsterdam A. The proteasome inhibitor MG132 promotes accumulation of the steroidogenic acute regulatory protein (StAR) and steroidogenesis. FEBS Lett. Feb. 9, 2001; 490(1-2):59-64.
18. Marambaud P; Ancolio K; Lopez-Perez E; Checler F, Proteasome inhibitors prevent the degradation of familial Alzheimer""s disease-linked presenilin 1 and potentiate A beta 42 recovery from human cells., Molecular Medicine 1998, v.4:147-157.
19. Gandy S; Naslund J; Nordstedt C, Alzheimer""s disease Molecular consequences of presenilin-1 mutation, Nature Jun. 7, 2001; 411 (6838): 654-5.
20. Russo C; Schettini G; Saido T C; Hulette C; Lippa C; Lannfelt L; Ghetti B; Gambetti P; Tabaton M; Teller J K, reply: Alzheimer""s disease Molecular consequences of presenilin-1 mutation, Nature Jun. 7, 2001; 411 (6838): 655
21. Russo C; Schettini G; Saido T C; Hulette C; Lippa C; Lannfelt L; Ghetti B; Gambetti P; Tabaton M; Teller J K, Presenilin-1 mutations in Alzheimer""s disease., Nature Jun. 1, 2000; 405 (6786): 531-2
22. Glenner G G; Wong C W, Alzheimer""s disease and Down""s syndrome: sharing of a unique cerebrovascular amyloid fibril protein., Biochem Biophys Res Commun Aug. 16, 1984; 122 (3): 1131-5.
SEQ ID NO:1 cDNA encoding mutant presenilin-1 (nucleotides 772-777 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:2 cDNA encoding mutant presenilin-1 (nucleotides 1156-1161 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:3 cDNA encoding mutant presenilin-1 (nucleotides 772-777 and 1156-1161 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:4 mutant presenilin-1 (amino acids 258-259 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s )
SEQ ID NO:5 mutant presenilin-1 (amino acids 386-387 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s)
SEQ ID NO:6 mutant presenilin-l (amino acids 258-259 and 386-387 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s)
SEQ ID NO:7 cDNA encoding mutant presenilin-2 (nucleotides 790-795 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:8 cDNA encoding mutant presenilin-2 (nucleotides 1099-1104 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:9 cDNA encoding mutant presenilin-2 (nucleotides 790-795 and 1099-1104 site directed mutagenesis sites directed by xe2x80x9cn""s)
SEQ ID NO:10 mutant presenilin-2 (amino acids 264-265 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s)
SEQ ID NO:11 mutant presenilin-2 (amino acids 367-368 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s)
SEQ ID NO:12 mutant presenilin-2 (amino acids 264-265 and 367-368 variable amino acids denoted by xe2x80x9cxxe2x80x9d""s)
SEQ ID NOS: 13-20 Mutagenesis oligonucleotides